Combination comprising immunostimulatory oligonucleotides

ABSTRACT

The invention relates to a combination and its use for the treatment of diseases. The instant disclosure provides a combination of a so-called T-cell regulator selected from the group comprising PD1, PD-L1, OX40, TIM-3, LAGS, CD137(4-1BB) and a non-codiung immuno-modulating DNA.

FIELD OF THE INVENTION

The invention relates to a combination and its use for the treatment ofdiseases.

BRIEF DESCRIPTION OF THE RELATED ART

The term “immunotherapy” defines the treatment of diseases bystimulating, inducing, enhancing or suppressing an immune response. Thestrategy of immunotherapies is to fight diseases, such as cancer,infectious diseases, allergy and asthma.

A variety of active agents, so called immunomodulators, that can be usedin immunotherapy are known. Most established immunomodulators belong tosmall molecules or nucleic acids, many of which interact with thetoll-like receptor system. Most known immunomodifying short DNAsequences contain an unmethylated cytosine guanine motif (CG motif),which has been described by Krieg et al. (Nature 1995 374: 6522546-549). The occurrence of unmethylated CG motifs is substantiallysuppressed in the genome of eukaryotes compared to prokaryotes orviruses. Therefore, DNA molecules containing such a motif have evolvedas a natural “danger signal” and trigger the immune system in the fightagainst prokaryotic or viral pathogens. This can be exploitedtherapeutically or prophylactically by using such sequences to treat orprevent infectious diseases with immunotherapy. A particular emphasishas in recent years been put on the use of such immunomodulators incancer therapy, with the aim of activating the patient's own immunesystem to fight against tumors.

DNA constructs comprising unmethylated CG motifs are able to elicit aconsiderable physiological effect by strongly stimulating effector cellsof the innate immune system including dendritic cells, macrophages,natural killer (NK) and NKT cells. Unmethylated CG motifs are detectedby the innate immune pattern recognition receptor Toll-like receptor(TLR) 9. While the exact recognition mechanism is not yet fullyunderstood, significant progress in unraveling the underlying pathwayshas been made (A. Krieg, Nat. Rev. Drug Disc., 5:471-484, 2006).

It is assumed that upon binding of DNA constructs containingunmethylated CGs to the receptor, multiple signal cascades are activatedin responding cells. By upregulation of characteristic surface moleculesand secretion of cytokines, adaptive immunity with a predominant Th1pattern is induced. Such constructs can be used in combination with, forexample, antibodies, chemotherapy or radiation therapy, vaccines orcytokines. Allergic diseases and asthma are mostly Th2-mediated. Byincreasing the ratio of Th1/Th2, the Th2-mediated responses areattenuated and thereby these types of diseases can be treated orprevented.

Surface molecules, which are unregulated by the TLR-9 pathway, include,for example, CD40, CD69, CD80, CD86 or CD169, depending on the celltype. The enhanced secretion of cytokines is also characteristic fordistinct cell types; cytokines include, for example, macrophageinflammatory proteins (MIP)-1 alpha, MIP-1beta, interleukin (IL)-6,IL-8, interferon (IFN)-alpha, tumor necrosis factor (TNF)-alpha,IFN-gamma, monocyte chemotactic protein (MCP)-1 or IFN-gamma-inducedprotein of 10 kDa (IP-10).

In order to prevent or treat diseases, vaccination has been proven as avery effective approach. To ensure a strong and durable immune response,adjuvants capable of stimulating antigen-presenting cells such asdendritic cells, are usually administered together with the antigen, andfor that purpose TLR9 agonists have been shown to be potentimmunostimulants.

Preclinical and ongoing clinical studies support the use of TLR-9agonists as immunomodulators and/or adjuvants, and prove theiranti-tumor effect by enhancing both the humoral and cellular responses.

Independently of any explanations of the underlying mechanisms by whichunmethylated CG motifs influence or modulate an immune response, manyapproaches were developed for modulation of the immune system by usingsuch motifs. The WO 1998/018810 discloses that immunostimulatorysequences containing unmethylated CG motifs are even more effective whenthey are part of a single strand. However, administering an open-chainedsingle-stranded DNA molecule is not practicable due to the quickdegradation of single-stranded nucleic acids. Consequently, differentmethods for the protection of single- or double-stranded DNA constructscomprising an unmethylated CG motif were developed.

To achieve resistance against the degradation by DNA nucleases thephosphodiester bonds in the backbone of a nucleic acid polymer arefrequently modified to phosphorothioates. Besides a somewhat lessstimulatory activity of such phosphorothioate-protected nucleic acidsclinical trials within the last years showed that the toxicity of aphosphorothioate-protection exclude or severely limit such nucleic acidsfrom any use in pharmaceutical compositions or medicaments.

From the four classes of known activators with distinct immunomodulationprofiles all members except two comprise linear DNA molecules. Oneexception is disclosed in EP 1 196 178. This document discloses shortdeoxyribonucleic acid molecules, comprising a partially single-stranded,dumbbell-shaped, covalently closed sequence of nucleotide residuescomprising CG motifs (“dSLIM”) consisting entirely of natural DNA.According to the disclosure of the EP 1 196 178 the CG motifs arelocated within the single-stranded loops at both ends of thedouble-stranded stem of the disclosed molecule or within thedouble-stranded stem. The single-stranded hairpin loops protect adouble-stranded stem from degradation by DNA nucleases within or outsideof the cell. GB 1402847.6 discloses a somewhat similar dumbbellstructure utilizing a different sequence.

Another exception from linear oligonucleotides is disclosed in WO2012/085291. This document teaches DNA constructs comprising nucleotidesin L-conformation. According to the data disclosed in WO 2012/085291,the number of nucleotides in L-conformation and their position withinthe DNA construct influences the immunostimulatory capability of the DNAconstruct. A DNA construct comprising only nucleotides in L-conformationdoes for instance not efficiently stimulate the immune system.

Document WO 2010/039137 discloses immune regulatory oligonucleotides asantagonists for TLR mediated diseases having one or more chemicalmodifications in the sequence flanking an immune stimulatory motifand/or in an oligonucleotide motif that would be immune stimulatory butfor the modification. Thus, the intention of the disclosedoligonucleotides of WO 2010/039137 is to suppress an immune responsecaused by TLRs.

WO 2005/042018 describes new so-called C-class CpG oligonucleotides,wherein a c-class oligonucleotide is characterised by CpG sequences,generally positioned at or near the 5′ end or 3′ end of the molecule,and a GC-rich palindrome motif, generally positioned at or near theother end of the molecule. The document discloses variations of thepalindromic sequence of a c-class DNA.

Document WO 2015/124614 discloses covalently closed DNA construct, apharmaceutical composition and a vaccine and their use for themodulation of the immune system, wherein the DNA construct comprisesspecific DNA sequences.

The strong stimulation of a cellular immune response makes it possibleto influence regulatory circuits, and without such intervention nosatisfactory immune activity would occur in the patient. This includesinducing a response to “weak” antigens, i.e. non-activating within MHC-Ipresentation, for example breakpoint peptides from chromosomaltranslocations or mutated oncogenes, often occurring in tumour diseases(Melief C J, Kast W M; T-cell immunotherapy of cancer; Res Immunol 1991June-August; 142(5-6):425-9; also: Pasternak G, Hochhaus A, SchultheisB, Hehlmann R; Chronic myelogenous leukemia: molecular and cellularaspects; J Cancer Res Clin Oncol 1998; 124(12):643-60). It may also bedesirable to break the tolerance to auto-antigens such as the tyrosinaseor tyrosinhydroxylase expressed in tumour cells of malignant melanomaand represented in MHC-I. (Weber L W, Bowne W B, Wolchok J D, SrinivasanR, Qin J, Moroi Y, Clynes R, Song P, Lewis J J, Houghton A N; Tumorimmunity and autoimmunity induced by immunization with homologous DNA; JClin Invest 1998 Sep. 15; 102(6):1258-64; Surman D R, Irvine K R,Shulman E P, Allweis T M, Rosenberg S A, Restifo N J; Generation ofpolyclonal rabbit antisera to mouse melanoma associated antigens usinggene gun immunization; Immunol Methods; 1998 May 1; 214(1-2):51-62).

Another, extremely important aspect is the adjuvant effect of ISS inprophylactic vaccinations as well as the possibility of re-polarizingthe reaction of an existing infection from a type-2 response to a type-1response, thus enabling the pathogen to be controlled (Kovarik J, et al.CpG oligodeoxynucleotides can circumvent the Th2 polarization ofneonatal responses to vaccines but may fail to fully redirect Th2responses established by neonatal priming; J Immunol. 1999 Feb. 1;162(3):1611-7). It has been demonstrated for a large number of pathogensthat the type of immune response has a decisive influence on the courseof the infection or on the patient's ability to survive. As far asallergic reactions represent a type-2 overshoot response, ISS isexpected to provide a therapeutic effect for indications of this kind aswell.

It has been observed that certain sequences containing CpGs possess acharacteristic which neutralises ISS-induced stimulation, i.e. thatsequences of this kind are able to suppress the stimulatory effect ofISS when added to them (Krieg A M, Wu T, Weeratna R, Efler S M,Love-Homan L, Yang L, Yi A K, Short D, Davis H L; Sequence motifs inadenoviral DNA block immune activation by stimulatory CpG motifs; ProcNatl Acad Sci USA 1998 Oct. 13; 95(21):12631-6). Without having fullyexplained the underlying mechanism of the effect of these sequencemotifs described as neutralising CpG motifs (“CpG-N”), the authors ofthe publication quoted here imply that this effect is limited toblocking stimulation by ISS. As long as the mechanism of immuneinduction by ISS is not explained, one cannot rule out the possibilitythat these CpG-N motifs also possess other immunomodifying properties oftherapeutical significance.

There is at least one human disease, systemic lupus erythematosus, whichis characterized by the confirmed existence of anti-DNA antibodies inpatient serum, and where it is suspected that a reaction to bacterialISS has aetiological reasons (Krieg A M, CpG DNA: a pathogenic factor insystemic lupus erythematosus?, J Clin Immunol 1995 November;15(6):284-92). In these cases and in other indications, blocking theunderlying mechanisms using CpG-N motifs would be beneficial.

Independent of any explanation of the underlying mechanisms, thepotential of CpG sequences for influencing the immune response isconsiderable and has generated sudden and widespread scientific interestin the phenomenon as well as in exploring the possibilities fortherapeutic and prophylactic applications where infections, tumours andimmune deficiencies are concerned.

The literature concerning ISS states (see e.g. WO09818810A1, p. 17, 1129-30), and this is confirmed by the invention described (see below),that immunostimulatory sequences containing CpGs are more effective whenthey occur as single strands. Administering short, open-chain,single-strand ISS oligodeoxynucleotides with the objective of immunemodification is the next logical step to take, and is the subject ofnumerous experimental approaches for treating infectious illnesses,tumours and autoimmune diseases. However, open-chain, single-strandoligodeoxynucleotides are degraded very quickly by extracellular andintracellular exonucleases and are therefore very difficult to use inin-vivo applications. The nucleases mentioned display considerablyreduced enzymatic activity when compared to modified phospho-ester bondsin the backbone of nucleic acid polymers; this has led to phosphorthioesters (“thioates”) or reduced phosphor bonds (phosphonates) inchiral or achiral form being used in applications where single-strandnucleic acid molecules are to be administered to the patient. Thesemodified compounds can be produced by solid phase synthesis, yet to someextent only by considerably more complicated methods by comparison withclassic DNA amidite synthesis. These compounds are known from antisenseresearch; in clinical studies of antisense strategies, however, it wasalso demonstrated that they have considerable side effects, particularlyon the blood coagulation system and complement system (see e.g. Sheehanand Lan, Blood 92, 1617-1625 (1998)). In connection with the use ofthiophosphoric acid derivatives for nuclease protection of ISS it wasalso demonstrated that the sequences display less stimulatory activitywhen those cytosine-guanosine residues which are actually effective arethemselves protected by the flanking sequences required for the activityitself (see WO 98/18810).

The teaching concerning use and production of immunostimulatory ISScontaining CpGs is comprehensively described in WO 98/18810, as well asin the documents quoted therein. The necessity for protectingoligodeoxynucleotides from exonucleases is described in detail in WO98/18810. A number of solutions are presented for solving the problem ofinsufficient in vivo stability, which are however expressly limited tosingle-strand linear ODNs; mention is made of thiophosphate esters,dithiophosphate esters or phosphonates. The possibility of stabilisingthe ODN by creating secondary structures, in particular a stem-loop, isnoted in WO 98/18810. Production and use of phosphorothioate oligomersin connection with immunostimulatory sequences is described in U.S. Pat.Nos. 5,663,153, 5,723,335 as well as in U.S. Pat. No. 5,856,462.

A different strategy for protecting single-strand sequences is describedin U.S. Pat. No. 5,750,669. Here the ends of the oligomers are linkedwith nucleoside residues connected by 5′-5′ and 3′-3′ bonds, which blockexonucleolytic degradation.

Double stem-loop or covalently closed, dumbbell-shaped ODNs are knownfrom experimental approaches in which competition in bonding sites forDNA-binding proteins, as well as transcription factors were the focus ofresearch (Lim et al. 1997, Nuc. Acids Res. 25, 575-581; Blumenfeld etal., Nuc. Acids Res. 1993, 21, 3405-3411).

The T cell response of the human immune system is regulated by multipleT-cell regulating molecules to avoid over-activation of the immunesystem on healthy cells (Pardoll D M. Nat Rev Cancer. 2012;12(4):252-264; Sharma P, Wagner K, Wolchok J D, Allison J P. Nat RevCancer. 2011; 11(11):805-812). Such T-cell regulating molecules aresummarized as “T-cell regulator” within the context of the instantdisclosure and comprise checkpoint inhibitors and co-stimulants. Tumorcells often take advantage of these regulatory systems to escapedetection by the immune system. Inhibition of a checkpoint of the immunesystem and co-stimulation of the T-cell system may enhance theanti-tumor immune response. The blockade of immune checkpoints and thusliberation of tumor-specific T cells to exert their effector functionagainst tumor cells has demonstrated efficacy in cancer settings, andclinical trials are ongoing (Hodi F S, O'Day S J, McDermott D F, et al.N Engl J Med. 2010; 363(8):711-723; Robert C, Thomas L, Bondarenko I, etal. N Engl J Med. 2011; 364(26):2517-2526; Wolchok J D, H. Kluger, M. K.Callahan, et al. N Engl J Med, 369 (2013), pp. 122-133).

Cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed cell death (PD)-1represent two checkpoints, which have been studied most extensively astargets for cancer therapy so far. CTLA-4 is a potent co-inhibitor thathas been shown to be aberrantly upregulated on the surface of T cells incertain cancers. It decreases T-cell activation in response to tumorcells and is thus involved in early T-lymphocyte tolerance. PD-1 hasbeen found to be upregulated in certain tumors, inhibiting T-cellfunction helping the tumor to evade the immune system by playing a rolein maintaining peripheral T-lymphocyte tolerance (Keir M E, Butte M J,Freeman G J, Sharpe A H, et al. Annu Rev Immunol. 2008; 26:677-704;Mahoney K M, Freeman G J, McDermott D F. Clinical Therapeutics 37(4):764-782, 2015).

The first immune-checkpoint inhibitor approved by the US Food and DrugAdministration (FDA) in 2011was ipilimumab, a monoclonal antibody thatblocks CTLA-4 for the treatment of metastatic melanoma. Blocking theinteraction between PD-1 and one of its ligands, PD-L1 (also known atB7-H1 and CD274), has been reported to generate antitumor responses(Pardoll D M. Nat Rev Cancer. 2012; 12(4):252-264).

Another inhibitory molecule, lymphocyte activation gene-3 (LAG-3), a CD4homolog that binds to MHC class II molecules, is expressed on activatedT cells, B cells, NK cells, and tumor-infiltrating lymphocytes, and isthought to negatively regulate T-cell expansion by limiting T-cellactivation (Pardoll D M. Nat Rev Cancer. 2012; 12(4):252-264; Goldberg MV, Drake C G. Curr Top Microbiol Immunol 2011;344:269-78). Its blockadeaugments T cell proliferation and enhances anti-tumor T cell responses(Nguyen L T, Nat Rev Immunol, 2015).

Further, T-cell immunoglobulin mucin-3 (TIM-3), the ligand of which isgalectin 9 (upregulated in various types of cancer), is expressed byIFN-secreting helper T (TH 1) cells, as well as dendritic cells,monocytes, and T cells [Ngiow S F, Teng M W, Smyth M J. Cancer Res.2011; 71(20:6567-71]. It inhibits T helper 1 cell responses, and TIM-3antibodies enhance antitumor immunity (Anderson A C. Curr Opin Immunol2012; 24:213-6). When bound to its ligand, galectin-9, TIM-3 induces TH1cell death (Zhu C, Anderson A C, Schubart A, et al. Nat Immunol. 2005;6(12):1245-52). Studies of TIM-3-deficient mice suggest that the TIM-3pathway inhibits the expansion and effector functions of TH 1 cells andmay be important for tolerance induction of TH1 cells (Sabatos C A,Chakravarti S, Cha E, et al. Nat Immunol. 2003; 4(11):1102-10). TIM-3has also been reported to be co-expressed with PD-1 on tumor-specificCD8+ T cells, and dual blockade of both molecules significantly enhancesthe in vitro proliferation and cytokine production of human T cells. Inanimal models, coordinate blockade of PD-1 and TIM-3 was reported toenhance anti-tumor immune responses and tumor rejection (Pardoll D M.Nat Rev Cancer. 2012; 12(4):252-264).

B- and T-lymphocyte attenuator (BTLA/CD272) was identified as aninhibitory receptor on T cells and. HVEM/TNFRSF14—which is expressed ontumor cells as well as on tumor-associated endothelial cells—was shownto be the BTLA ligand. BTLA expression levels are high on tumorinfiltrating lymphocytes (TIL) from patients with melanoma andBTLA-expressing T cells are inhibited in the presence of its ligand,HVEM. BTLA can inhibit the function of tumor-specific human CD8⁺ T cells(Paulos C M, June C H. J Clin Invest 2010; 120:76-80). Thus, BTLA mayalso be a relevant inhibitory receptor for T cells in the tumourmicroenvironment and a target for checkpoint inhibition strategies(Pardo11 D M. Nat Rev Cancer. 2012; 12(4):252-264).

OX40 (CD134/TNFRSF4) is a member of the TNFR super-family and isexpressed by CD4 and CD8 T cells during antigen-specific priming.Ligation of OX40 on CD8 and CD4 T cells promotes their survival andexpansion. Furthermore activating OX40 boosts the generation oftumor-reactive effector T cells and inhibits T-cell function.Preclinical studies demonstrated that treatment of tumor-bearing hostswith OX40 agonists resulted in tumor regression in several preclinicalmodels (Linch S N, McNamara M J, Redmond W L. Front Oncol. 2015 5:34).

The co-stimulatory receptor CD137 (4-1BB/TNFSF9) possesses an unequaledcapacity for both activation and pro-inflammatory polarization ofanti-tumor lymphocytes. Co-stimulation through the CD137/4-1BB receptoractivates multiple signaling cascades within the T cell, powerfullyaugmenting T cell activation. Stimulation of CD137 on antigen-primedT-lymphocytes increase tumor immunity and CD137 monotherapy is capableof mediating significant tumor regressions and even cures of numeroustypes of established murine tumors (Bartkowiak T, Curran M A. FrontOncol. 2015 5:117).

Based on this state of the art, the objective of the instant disclosureis to provide an efficient combination comprising immunostimulatory DNAconstructs and its use as a medicament.

SUMMARY OF THE INVENTION

With regard to the prior art it is an objective of the instantdisclosure to provide a combination of molecule binding to a T-cellregulator and an immunomodulating DNA construct in form of a non-codingsequence of deoxyribonucleotides.

The present disclosure teaches a combination comprising the componentsof a chemical or molecule binding to at least one of the moleculesselected from the group comprising PD1, PD-L1, OX40, TIM-3, LAGS,CD137(4-1BB) for affecting their function as checkpoint inhibitors orco-stimulants; and a non-coding sequence of deoxyribonucleic acidscomprising at least one sequence motif N¹N²CGN³N⁴, wherein N is anucleotide comprising A, C, T, or G, and C is deoxycytidine, G isdeoxyguanosine, A is deoxyadenosine and T is deoxythymidine.

The molecule binding to a T-cell regulator may be a protein or peptide,like an antibody, which is synthetically or biologically manufactured.

The combination of the instant disclosure may comprise for N¹N² anelement taken from the group of GT, GG, GA, AT and AA, and for N³N⁴ anelement taken from the group of CT, TG and TT.

The non-coding sequence of deoxyribonucleic acids may either be linearopen-chained on both sides, linear open-chained on one side of a doublestranded part with a single stranded hairpin on the respective otherside of the double strand or a dumbbell-shaped partially single-strandedcovalently closed chain of deoxyribonucleic acids.

The combination may further comprise at least three of said sequencemotifs N¹N²CGN³N⁴.

It is intended for a linear open-chained non-coding sequence ofdeoxyribonucleic acids that it may comprise at least one nucleotide inL-conformation, wherein one of the five terminal nucleotides located atthe 5′- and/or the 3′-end of a DNA single strand of the linearopen-chained non-coding sequence of deoxyribonucleic acids may be inL-conformation.

The combination of the instant disclosure may further comprise at leastone of the following non-coding sequences of deoxyribonucleotides

a. (SEQ ID NO: 1) GTTCCTGGAG ACGTTCTTAG GAACGTTCTC CTTGACGTTG GAGAGAAC;or b. (SEQ ID NO: 2) ACCTTCCTTG TACTAACGTT GCCTCAAGGA AGGTTGATCTTCATAACGTT GCCTAGATCA, or c. (SEQ ID NO: 3) AACGTTCTTCGGGG CGTT, or d.(SEQ ID NO: 4) AGGTGGTAAC CCCTAGGGGT TACCACCTTC ATCGTCGTTTTGTCGTTTTG TCGTTCTT.

The combination may further comprise a non-coding sequence ofdeoxyribonucleic acids with a length of 40 to 200 nucleotides or of 48to 116 nucleotides.

It is further intended that the sequence AACGTTCTTCGGGG CGTT (SEQ IDNO:3) may be part of the sequence CCTAGGGGTT ACCACCTTCA TTGGAAAACGTTCTTCGGGG CGTTCTTAGG TGGTAACC CCTAGGGGTT ACCACCTTCA TTGGAAAACGTTCTTCGGGG CGTTCTTAGG TGGTAACC (SEQ ID NO:5).

The sequence motif N¹N²CGN³N⁴ can be part of a single stranded region ofa non-coding sequence of deoxyribonucleotides, which is part of acombination according to the instant disclosure.

The combination may provide the components of both groups in a solid,liquid or gaseous form to be applied with maximal 15 mg/kg weight. Thismeans that the dosage can be adapted to the weight of the organism towhich the combination should be applied.

A method comprising the step of providing the components of acombination of the instant disclosure simultaneously, alternating orsuccessively is another object of the invention. The non-coding sequenceof deoxyribonucleic acids may be provided prior to the chemical ormolecule for affecting the T-cell regulator or vice versa.

A further object of the instant disclosure is the use of the disclosedcombination as a medicament or for the treatment of diseases likecancer, autoimmune diseases and inflammation.

The compounds of the disclosed combination may be administeredsimultaneously, alternating or successively for the treatment of cancer,autoimmune diseases and inflammation.

A use of the disclosed combination for the manufacture of apharmaceutical or pharmaceutically preparation, including vaccines,comprising acceptable pharmaceutical salts is a further object of theinstant invention. The pharmaceutical may release the compounds of thedisclosed combination simultaneously, alternating or successively.

Finally, the use of a combination of the instant disclosure as anadjuvant in therapeutic or prophylactic vaccination is an object of theinstant disclosure.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described on the basis of figures. It will beunderstood that the embodiments and aspects of the invention describedin the figures are only examples and do not limit the protective scopeof the claims in any way. The invention is defined by the claims andtheir equivalents. It will be understood by the skilled artisan thatfeatures of one aspect or embodiment of the invention can be combinedwith a feature of a different aspect or aspects of other embodiments ofthe invention. It shows:

FIG. 1A, B Anti-tumor activity of the combination of SEQ ID NO:5 withanti-PD-1.

FIG. 2 In vitro stimulation of human PBMC with peptides selected fromHLA class I-restricted T-cell epitopes of recall-antigens.

FIG. 3A, B Anti-tumor activity of the combination of SEQ ID NO:5 withanti-PD-L1.

FIG. 4A, B Anti-tumor activity of the combination of SEQ ID NO:6 withanti-PD-1 .

FIG. 5 In vitro stimulation of human PBMC with peptides selected fromHLA class I-restricted T-cell epitopes of recall-antigens, SEQ ID NO:6and anti-PD-.

FIG. 6A, B Anti-tumor activity of the combination of SEQ ID NO:7 withanti-CTLA-4.

FIG. 7A, B Anti-tumor activity of the combination of SEQ ID NO:7 withanti-PD-L1.

FIG. 8A-C In vitro stimulation of human PBMC with peptides selected fromHLA class I-restricted T-cell epitopes of recall-antigens, SEQ ID NO:9and anti-PD-.

FIG. 9A, B Anti-tumor activity of the combination of SEQ ID NO:10 withanti-PD-1.

FIG. 10A, B Anti-tumor activity of the combination of SEQ ID NO:10 withanti-CTLA-4.

FIG. 11 In vitro stimulation of human PBMC with peptides selected fromHLA class I-restricted T-cell epitopes of recall-antigens, SEQ IDNO:10and anti-PD-.

FIG. 12 In vitro stimulation of human PBMC with peptides selected fromHLA class I-restricted T-cell epitopes of recall-antigens, SEQ ID NO:11and anti-PD-1.

FIG. 13 In vitro stimulation of human PBMC with peptides selected fromHLA class I-restricted T-cell epitopes of recall-antigens, EnanDIM362and anti-PD-1.

DETAILLED DESCRIPTION OF THE INVENTION

The instant invention provides a combination of a molecule binding to aso called T-cell regulator and a non coding sequence of deoxyribonucleicacids.

Within the meaning of the present disclosure a linear open-chained DNAsequence is designated as oligonucleotide, abbreviated with ODN. SaidDNA sequence can be single-stranded or partially or completelydouble-stranded. The terms oligo, oligonucleotide andoligodeoxynucleotide are used synonymously and do not indicate alimitation of the length of the corresponding DNA sequence. The singlecomponents of oligonucleotides are nucleotides.

An oligo can be manufactured synthetically or be partially or completelyof biological origin, wherein a biological origin includes geneticallybased methods of manufacture of DNA sequences.

L-DNA or nucleotides in L-conformation refer to nucleotides, whichcomprises L-deoxyribose as the sugar residue instead of the naturallyoccurring D-deoxyribose. L-deoxyribose is the enantiomer (mirror-image)of D-deoxyribose. Oligonucleotides partially or completely consisting ofnucleotides in L-conformation can be partially or completely single- ordouble-stranded; however, nucleotides in L-conformation cannot hybridizeto nucleotides in D-conformation (Hauser et al., Nucleic Acid Res. 200634: 5101-11). L-DNA is equally soluble and selective as D-DNA. Yet,L-DNA is resistant towards enzymatic exoactivity of naturally occurringenzymes, especially exonucleases, so L-DNA is protected againstintracellular degradation (Urata et al., Nucleic Acids Res. 1992 20:3325-32). Therefore, L-DNA is very widely applicable.

A “stem” according to the present disclosure shall be understood as aDNA double strand formed by base pairing either within the sameoligonucleotide (which is then partially self-complementary) or withindifferent oligonucleotides (which are partially or completelycomplementary). Intramolecular base-pairing designates base-pairingwithin the same oligonucleotide and base-pairing between differentoligonucleotides is termed as intermolecular base-pairing.

A “loop” within the meaning of the present disclosure shall beunderstood as an unpaired, single-stranded region either within or atthe end of a stem structure. A “hairpin” is a distinct combination of astem and a loop, which occurs when two self-complementary regions of thesame oligonucleotide hybridize to form a stem with an unpaired loop atone end.

A “solid phase” to which the nucleotides are covalently ornon-covalently attached refers to, but is not restricted to, a column, amatrix, beads, glass including modified or functionalized glass, silicaor silica-based materials including silicon and modified silicon,plastics (comprising polypropylene, polyethylene, polystyrene andcopolymers of styrene and other materials, acrylics, polybutylene,polyurethanes etc.), nylon or nitrocellulose, resins, polysaccharides,carbon as well as inorganic glasses and plastics. Thus, microtiterplates are also within the scope of a solid phase according to thepresent disclosure.

Immunomodulation according to the present disclosure refers toimmunostimulation and immunosuppression. Immunostimulation meanspreferentially that effector cells of the immune system are stimulatedin order to proliferate, migrate, differentiate or become active in anyother form. B cell proliferation for instance can be induced withoutco-stimulatory signals by immunostimulatory oligonucleotides, whichnormally require a co-stimulatory signal from helper thymocytes.

Immunosuppression on the other hand shall be understood as reducing theactivation or efficacy of the immune system. Immunosuppression isgenerally deliberately induced to prevent for instance the rejection ofa transplanted organ, to treat graft-versus-host disease after a bonemarrow transplant, or for the treatment of autoimmune diseases such as,for example, rheumatoid arthritis or Crohn's disease.

In this context, immunomodulation may also refer to the influence of thenature or the character of an immune reaction, either by affecting ormodifying an immune reaction, which is still developing or maturing orby modulating the character of an established immune reaction. Thus,affecting means in the context of checkpoint inhibitors to suppresstheir inhibitory effect, and in the context of costimulatory moleculesto activate them.

The term “cancer” comprises cancerous diseases or a tumor being treatedor prevented that is selected from the group comprising mammarycarcinomas, melanoma, skin neoplasms, gastrointestinal tumors, includingcolon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer,small intestine cancer, ovarial carcinomas, cervical carcinomas, lungcancer, prostate cancer, kidney cell carcinomas and/or liver metastases.

Autoimmune diseases according to the present disclosure compriserheumatoid arthritis, Crohn's disease, systemic lupus (SLE), autoimmunethyroiditis, Hashimoto's thyroiditis, multiple sclerosis, Graves'disease, myasthenia gravis, celiac disease and Addison's disease.

An agonist within the meaning of the instant disclosure and inaccordance with its common definition represents a chemical or moleculethat binds to another molecule, like a receptor or ligand and thusactivates the molecule. In contrast to an agonist that activates, anantagonist shall be understood as a chemical or molecule that blocks theinteraction of the molecule to which the antagonist binds with arespective agonist. Depending on the context, an antagonist in theunderstanding of the instant invention may also result in the activationof a process, because the antagonist blocks the interaction of anotherantagonist with a receptor for instance.

The term “pharmaceutically applicable or acceptable salts” as usedherein includes salts of a compound of the combination, which areprepared with relatively nontoxic (i.e. pharmaceutically acceptable)acids or bases, depending on the particular substituents found on thecompounds of the present invention. If, for example, compounds of thepresent invention contain acidic functionalities, base addition saltsmay be obtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Non-limiting examples of pharmaceutically acceptable baseaddition salts include sodium, potassium, calcium, ammonium, organicamino, or magnesium salt, or a similar salt. If compounds of the presentinvention contain basic functionalities, acid addition salts may beobtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Non-limiting examples of pharmaceutically acceptable acidaddition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, phosphoric, partiallyneutralized phosphoric acids, sulfuric, partially neutralized sulfuric,hydroiodic, or phosphorous acids and the like, as well as the saltsderived from relatively nontoxic organic acids like acetic, propionic,isobutyric, maleic. malonic, benzoic, succinic, suberic, fumaric,mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention may contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts. Contacting the salt with a base mayregenerate the neutral forms of the compounds of the present inventionor acid and isolating the parent compound in the conventional manner.The parent form of the compound differs from the various salt forms incertain physical properties, such as solubility in polar solvents, butotherwise the salts are equivalent to the parent form of the compoundfor the purposes of the present invention. The compounds of the presentinvention may possess chiral or asymmetric carbon atoms (opticalcenters) and/or double bonds. The racemates, diastereomers, geometricisomers and individual optical isomers are encompassed by the presentinvention. The compounds of the present invention may exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms are equivalent to unsolvated forms and arealso encompassed by the present invention. The compounds of the presentinvention may furthermore exist in multiple crystalline or amorphousforms.

Deoxyribonucleic acid molecules, consisting of a partiallysingle-stranded, dumbbell-shaped, covalently closed chain ofdeoxyribonucleoside residues, which contain one or more sequences of thebase sequence N¹N²CGN³N⁴, wherein N¹N² is an element from the GT, GG,GA, AT or AA group, N³N⁴ is an element from the CT or TT group, as wellas C deoxycytosine, G deoxyguanosine, A deoxyadenosine and Tdeoxythymidine, are used in combination with chemicals or molecules ableto bind T-cell regulator of the immune system for immunostimulation inhumans or higher animals.

The deoxyribonucleic acid molecules relating to the instant disclosuremay have a length of up to 200 nucleotides. In particular, sequenceswith a length between 48 and 116 nucleotide are intended.

The dumbbell-shaped non-coding sequences of deoxyribonucleic acidmolecules may comprise the base sequence N¹N²CGN³N⁴ is theirsingle-stranded regions.

The immunostimulation may take place in vitro or in vivo.

The instant disclosure provides also linear open-chained DNA sequencecomprising at least one CpG motif and at least one nucleotide inL-conformation. Due to the partial/complete L-conformation, the DNAsequence has no 5′- or 3′-ends that are accessible to exonucleases. Incase that the construct has on one end of a double strand a singlestranded-loop, the end is also protected against degradation. Thereby,the ODNs are in total protected against cellular degradation withouthaving the need to use a phosphorothioate backbone, which has been shownto be toxic. In addition, the ODNs only consist of a minimum number ofnucleotides, which makes them small and thereby easy to transfect intocells.

The non-coding sequence of ddeoxyribonucleic acids comprising at leastone sequence motif N¹N²CGN³N⁴ can be single-stranded or partially orcompletely double-stranded. This includes base-pairing within the samemolecule (intramolecular) or within different molecules (intermolecular)or any combination thereof. It is also possible that the constructcomprises at least one unpaired, single-stranded region. As a furtherembodiment, hairpin structures are included. Due to the partial orcomplete L-conformation, a longer half-life of the construct is ensuredas nucleotides in L-conformation are not subject to degradation.

It is also within the scope of the instant disclosure that at least twomolecules, which are single-stranded or partially or completelydouble-stranded can ligate to each other to form multimeric constructs.These multimeric constructs thus incorporate at least as many CpG motifsas ligation partners, tightly packed within one molecule, and aretherefore expected to elicit also a considerable immune response as partof the combination with T-cell regulators. The resulting single-strandedor partially or completely double-stranded multimeric constructs caneither be covalently closed comprising nucleotides in L-conformationwithin the molecule or open multimeric constructs comprising nucleotidesin L-conformation at the 5′- and/or the 3′-end for protection againstcellular degradation.

The disclosure further comprises chemical modifications of at least onenucleotide in the non-coding sequence of deoxyribonucleic acidscomprising at least one sequence motif N¹N²CGN³N⁴ with a functionalgroup selected from the group comprising carboxyl, amine, amide,aldimine, ketal, acetal, ester, ether, disulfide, thiol and aldehydegroups. This allows coupling of the DNA construct to a compound selectedfrom the group comprising peptides, proteins, carbohydrates, antibodies,synthetic molecules, polymers, micro projectiles, metal particles or asolid phase by, for example, adsorption, covalent or ionic bonding.

The modification can be specifically selected for the respectivepurpose. The construct can thus be used, for example, to shuttle othermolecules to the specific cell responding to the CpG motif/sincorporated. In addition, it is possible by such modifications tocouple the construct to micro projectiles, which can be used to transferthe construct into the cell. The construct can also be coupled to asolid phase, e. g. a microtiter plate.

Experiments described below were performed to investigate the influenceof combining non-coding sequences of deoxyribonucleic acids with T-cellregulators. The experiments were conducted using dumbbell-shapedcomprising the sequence motif N¹N²CGN³N⁴, linear open-chained non-codingsequence of deoxyribonucleic acids comprising N¹N²CGN³N⁴, wherein thoseconstructs comprise nucleotides in L-conformation to prevent them fromdegradation. In addition, the effect of combining T-cell regulators witha non-coding sequence of deoxyribonucleic acids comprising N¹N²CGN³N⁴and twice the sequence of SEQ ID NO:4 were be investigated.

T-cell regulators antibodies binding to PD1, PD-L1, OX40, LAG-3, TIM3and CD137(4-1BB) were used in a mouse model with injected human tumors.The effect on therapy after a growth phase is described in more detailbelow on growth of tumors in comparison to control groups.

The experiments compare dosage regimen with simultaneous, alternating orsuccessive application of the components of the combination of theinstant disclosure. In addition to the qualitative application of thecompounds it was investigated whether reduced amounts of T-cellregulator are necessary for achieving comparable or even better resultsin applying the checkpoint inhibitor without a non-coding DNA sequencecomprising a N¹N²CGN³N⁴ sequence motif.

The in vitro analysis of the combinatory potential of TLR9 agonists withmolecules binding to T-cell regulators comprises the use of in vitrocell culture system of human PBMC for evaluation of their T cellsresponses after stimulation. Stimulation of PBMC will be achieved with amixture of immunogenic peptides from CMV, EBV, influenza andtetanus-toxin in the presence of antibodies against immunological T-cellregulators (e.g. PD-1, PD-L1, etc.) and TLR9 agonists (i.e. SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:6).

The quantification of cytokines (IL-2 and IFN-gamma) in cell culturesupernatants was determined. Although this in vitro cell culture systemcannot mirror the complex interactions of immune cells in vivo, itprovides evidences for an advantage of the combination of those TLR9agonists.

DETAILED DESCRIPTION OF THE FIGURES

The combination of SEQ ID NO:5 with anti-PD-1 showed a surprisinglyvastly increased anti-tumor effect compared to either anti-PD-1 or SEQID NO:5 monotherapy in a mouse A20 tumor model.

Tumor growth was surprisingly nearly completely inhibited by thecombination of SEQ ID NO:5 and PD-1 (FIG. 1A, B). 9-12 mice per groupwere inoculated s.c. with A20 murine tumor cells and injected with SEQID NO:5 (250 μg/application, i.tu. on day 14, 16, 19, 21, 23, 26, 28,30, 33 and 35), anti-PD-1 (100 μg/application i.p. on day 8, 11, 16 and19), or both. Injection of vehicle (i.tu.) served as control. FIG. 1Ashows the mean tumor growth-inlay, mean tumor growth inhibition from day18 to 32 (at day 29: 46.0% for SEQ ID NO:5, 54.2% for anti-PD-1, 99.9%for the combination). FIG. 1B shows a Kaplan-Meier survival plot.

The synergistic combinatory effect of SEQ ID NO:5 with anti-PD-1 shownin FIG. 1 A, B was confirmed in vitro when human peripheral bloodmononuclear cells (PBMC) were incubated with antigenic peptides and acombination of SEQ ID NO:5 and anti-PD-1. Peptides were selected fromHLA class-I-restricted T-cell epitopes of recall antigens (CMV, EBV,Flu=CEF) and the combination with SEQ ID NO:5 clearly increased theINF-gamma secretion by the PBMC compared to injection of SEQ ID NO:5 oranti-PD-1 alone (FIG. 2). The final concentration of the peptides was 1μg/ml per peptide, SEQ ID NO:5 was used in a concentration of 3 μM andanti-PD-1 with 10 μg/ml (n=4). IFN-gamma secretion was analyzed as amarker for immune response; normalized to IFN-gamma level afterstimulation of PBMC with CEF-peptides alone. Murine IgG (10 μg/ml) wasused as control for anti-PD-1.

Furthermore, in a mouse CT26 tumor model, the surprising beneficialeffects of the combination therapy of anti-PD-L1 and SEQ ID NO:5 wasalso clearly superior to the either SEQ ID NO:5 or anti-PD-L1monotherapy. Tumor growth was reduced (FIG. 3A) and survival wasaugmented (FIG. 3B). 10 mice per group were inoculated s.c. with CT26murine tumor cells and injected with SEQ ID NO:5 (250 μg/application,s.c. on day 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, and 26), anti-PD-L1 (10mg/kg per application, i.p. on day 3, 5, 7, 9, 11, 13, 15, 17), or both.Injection of vehicle (s.c.) served as control. FIG. 3A schows mean tumorgrowth-inlay, mean tumor growth inhibition from day 17 to 27 (at day 20:23.0% for lefitolimod, no inhibition for anti-PD-L1, 39.9% for thecombination). FIG. 1B shows a Kaplan-Meier survival plot.

The combinatorial effect of applying SEQ ID NO:6 having the the loopsequence TCATCGTCGTTTTGTCGTTTTGTCGTTCTT was also investigated.

SEQ ID NO:6 was administered together with anti-PD-1 in a mouse CT26tumor model. This combination surprisingly profoundly augmented theanti-tumor effect compared to the monotherapy with the single agents,SEQ ID NO:6 or anti-PD-1 (FIG. 4A, B). Again, 10 mice per group wereinoculated s.c. with CT26 murine tumor cells and injected with SEQ IDNO:6 (250 μg/application, i.tu. on day 15, 17, 19, 22, 24, 26, 29, 31),anti-PD-1 (100 μg/application, i.p. on day 3, 6, 10 and 13), or both.Injection of vehicle (i.tu.) served as control. FIG. 4A shows the meantumor growth-inlay, mean tumor growth inhibition from day 15 to 29 (atday 23: 48.2% for SEQ ID NO:6, no inhibition for anti-PD-1, 75.4 for thecombination). FIG. 1B shows a mean Kaplan-Meier survival plot.

The results shown in FIG. 4 are in line with the in vitro stimulationdata of human PBMC with antigenic peptides, also showing surprisingly abenefit of the combination over the single use of the compounds (FIG.5). Peptides selected from HLA class I-restricted T-cell epitopes ofrecall-antigens (CMV, EBV, Flu=CEF) with a final concentration of 1μg/ml per peptide, SEQ ID NO:6 in a concentration of 3 μM and anti-PD-1with 10 μg/ml (n=4) were used. IFN-gamma secretion was used as a markerfor immune response; normalized to IFN-gamma level after stimulation ofPMBC with CEF-peptides alone. Murine IgG (10 μg/ml) was used as controlfor anti-PD-1.

Oligos comprising nucleotides in L-conformation were used in furtherstudies. Those oligos comprise L-nucleotides at indicated positions. DNAmolecules were used with the core sequence [yTCATTxCGTGACGTGACGTTCzv](y=2 to 8 G, protected with 1 to 3 L-deoxyribose or not; x=3 to 4 A; z=2to 6 T protected with 1 to 3 L-deoxyribose; v=A, G, C, T).

These L-nucleotide comprising molecules showed increased immunemodulatory and anti-tumor properties when combined with checkpointinhibitors. For instance, combination of SEQ ID NO:7 (GGGGTCATTAAAACGTGACGTGACGTTCTTTTT, L-deoxyribose containing bases underlined)with anti-CTLA-4 in a mouse CT26 tumor model resulted in a surprisinglyefficient decreased tumor growth compared to SEQ ID NO:7 or anti-CTLA-4monotherapy (FIG. 6). 10 mice per group were inoculated s.c. with CT26murine tumor cells and injected with SEQ ID NO:7 (200 μg/application,s.c. on day 3, 5, 8, 10, 12, 15, 17, 19, 22, 14 and 26), anti-CTLA-4(100 μg/application at day 8; 50 μg/application at day 11 and 14, i.p.),or both. Injection of vehicle (s.c.) served as control. FIG. 6A showsthe mean tumor growth-inlay, mean tumor growth inhibition from day 15 to30 (at day 22: 19.8% for SEQ ID NO: 7, 59.1% for anti-PD-1, 65.3% forthe combination). FIG. 6B shows a Kaplan-Meier survival plot.

The combination of SEQ ID NO:7 with anti-PD-L1 also showed a moderatelyincreased anti-tumor effect compared to that of the single compounds SEQID NO:7 or anti-PD-L1 in the mouse CT26 tumor model (FIG. 7). 10 miceper group were inoculated s.c. with CT26 murine tumor cells and injectedwith SEQ ID NO:7 (s.c. on day day 3, 5, 7, 10, 12, 14, 17, 19, 21, 24,and 26), anti-PD-L1 (10 mg/kg per application, i.p. on day 3, 5, 7, 9,11, 13, 15, 17), or both. Injection of vehicle (s.c.) served as control.FIG. 7A shows the mean tumor growth-inlay, mean tumor growth inhibitionfrom day 13 to 27 (at day 20: 16.3% for SEQ ID NO:7, no inhibition foranti-PD-L1, 33.3% for the combination). FIG. 7B shows a Kaplan-Meiersurvival plot.

In in vitro studies the benefit of the combination of anti-PD-1 with SEQID NO:7, SEQ ID NO:8 (GGGGGGGGTCATTAAAACGTGACGTGACGTTCTTTTT,L-deoxyribose containing bases underlined), and SEQ ID NO:9(GGGGTCATTAAACGTGACGTGA CGTTCTTTTT, L-deoxyribose containing basesunderlined) was observed regarding IFN-gamma secretion from PBMCstimulated with antigenic peptides (FIG. 8). Peptides were selected fromHLA class I-restricted T-cell epitopes of recall-antigens (CMV, EBV,Flu=CEF; final concentration 1 μg/ml per peptide), SEQ ID NO:7 (A,n=12), SEQ ID NO:8 (B, n=2), SEQ ID NO:9 (C, n=4), each DNA molecule ata final concentration of 3 μM; and anti-PD-1 (10 μg/ml). Analysis ofIFN-gamma secretion as marker for immune response; normalized toIFN-gamma level after stimulation with CEF-peptides alone. Murine IgG(10 μg/ml ) was used as control for anti-PD-1

In another series of experiments DNA molecules with the core sequence[yTCATTxCGTTCTTCGGGGCGTTCzv] (y=2 to 8 G, protected with 1 to 3L-deoxyribose or not; x=3 to 4 A; z=2 to 6 T protected with 1 to 3L-deoxyribose; v=A, G, C, T) were used.

The combinatory effect regarding immunomodulation and anti-tumor effectwas established for this group as well. As example for this group, SEQID NO:10 (GGGGTCATTAAACGTTCTTCGGGG CGTTCTTTTT, L-deoxyribose containingbases underlined) was used to investigate the combination withanti-PD-1.

The combination resulted in a profound reduction of the tumor growth ina mouse CT26 tumor model (FIG. 9). 10 mice per group were inoculateds.c. with CT26 murine tumor cells and injected with SEQ ID NO:10 (200μg/application, s.c. on day 3, 5, 8, 10, 12, 15, 17, 19, 22, 14 and 26),anti-PD-1 (200 μg/application, i.p. on day 3, 6, 10 and 14), or both.Injection of vehicle (s.c.) served as control. FIG. 9A shows the meantumor growth-inlay, mean tumor growth inhibition from day 14 to 32 (atday 25: 17.8% for SEQ ID NO:10, 51.9% for anti-PD-1, 74.6% for thecombination). FIG. 9B shows a Kaplan-Meier survival plot.

In addition, a combination of SEQ ID NO:10 with anti-CTLA-4 lead to adecreased tumor growth in mouse CT26 tumor model compared to treatmentwith the single agents, SEQ ID NO:10 or anti-CTLA-4 (FIG. 10). 10 miceper group were inoculated s.c. with CT26 murine tumor cells and injectedwith SEQ ID NO:10 (200 μg/application, s.c. on day 3, 5, 8, 10, 12, 15,17, 19, 22, 14 and 26), anti-CTLA-4 (100 μg/application at day 8, 50μg/application at day 11 and 14, i.p.), or both. Injection of vehicle(s.c.) served as control. FIG. 10A shows the mean tumor growth-inlay,mean tumor growth inhibition from day 15 to 30 (at day 22: 49.7% for SEQID NO:10, 59.1% for anti-PD-1, 70.3% for the combination). FIG. 10Bshows a Kaplan-Meier survival plot.

Furthermore, a combination of SEQ ID NO:10 and anti-PD-1 was evaluatedin in vitro PBMC stimulation studies and showed an increased effectregarding IFN-gamma secretion compared to SEQ ID NO:10 or anti-PD-1alone (FIG. 11). Peptides were selected from HLA class I-restrictedT-cell epitopes of recall-antigens (CMV, EBV, Flu=CEF; finalconcentration 1 μg/ml per peptide)), SEQ ID NO:10 (3 μM) and anti-PD-1(10 μg/ml) (n=5). Analysis of IFN-gamma secretion served as marker foran immune response; normalized to IFN-gamma level after stimulation withCEF-peptides alone. Murine IgG (10 μg/ml) was used as control foranti-PD-1.

In a further experiment, a DNA molecule with a core sequence[yTCATTxTCGTCGTTTTGTCGTTTTGTCGzv] (y=2 to 8 G, protected with 1 to 3L-deoxyribose or not; x=3 to 4 A; z=2 to 6 T protected with 1 to 3L-deoxyribose; v=A, G, C, T) was used in experiments.

SEQ ID NO:11 (GGGGTCATTAAATCGTCGTTTTGTCGTTTTGTCGTTTTT, L-deoxyribosecontaining bases underlined) was used as example for this group. WhenSEQ ID NO:11 was combined with anti-PD-1 in vitro in PBMCstudies,—IFN-gamma secretion surprisingly significantly increasedshowing an improvement compared to SEQ ID NO:11 or anti-PD-1 alone (FIG.12). Peptides selected from HLA class I-restricted T-cell epitopes ofrecall-antigens (CMV, EBV, Flu=CEF; final concentration 1 μg/ml perpeptide), SEQ ID NO:11 (3 μM) and anti-PD-1 (10 μg/ml) (n=4). Analysisof IFN-gamma secretion as marker for immune response; normalized toIFN-gamma level after stimulation with CEF-peptides alone. Murine IgG(10 μg/ml) was used as control for aPD-1.

Finally, a DNA molecules with the core sequence [yACGATCGTCwT] (y=2 to 8G, protected with 1 to 3 L-deoxyribose or not; w=4 to 12 G protectedwith 1 to 3 L-deoxyribose) was used for testing effects of combinatorialapplication.

An example for this group is SEQ ID NO:12 (GGGGGACGATCGTCGGGGGGT,L-deoxyribose containing bases underlined). In in vitro stimulationstudies with human PBMC its combination with anti-PD-1 was evaluatedleading to a significantly augmented immune response compared to thesingle compounds (FIG. 13). Peptides were selected from HLA classI-restricted T-cell epitopes of recall-antigens (CMV, EBV, Flu=CEF;final concentration 1 μg/ml per peptide), SEQ ID NO:12 (3 μM) andanti-PD-1 (10 μg/ml) (n=4). Analysis of IFN-gamma secretion as markerfor immune response; normalized to IFN-gamma level after stimulationwith CEF-peptides alone. Murine IgG (10 μg/ml) was used as control foraPD-1.

Taking the above described experimental settings into account and aweight of a mouse of about 20 g, the amounts of DNA to be applied liesin a range of about 12.5 mg/kg weight, so that it seems to be feasiblethat maximal 15 mg/kg will be necessary for obtaining the shownsurprising results.

Anti-PD-1 antibody has been applied with 10 mg/kg weight so that theapplication of maximal 15 mg/kg weight also seems to be necessary forobtaining the shown surprising results.

1-22. (canceled)
 23. A linear open-chained non-coding sequence ofdeoxyribonucleic acids selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9 and SEQ ID NO: 11.